1. Field of the Invention
Present invention relates to the applications of surfactin, in particular, applications of surfactin in cosmetic products.
2. Description of the Prior Art
1. Human Skin Structure and its Aging Factors
Skin is the major organ on the surface in the human body and its thickness varies with the location, age, and gender. The main function of skin is to serve as the primary barrier to prevent damage to the internal organs by external environmental factors, e.g. UV light, temperature variations, humidity changes and particulate matter and viral or bacterial entry. Therefore, the aging mechanism can be easily triggered in the skin by external factors to replace damaged cells.
(1) Cellular Aging and Genetic Defects
Aging starts to take effect on cells followed by tissues and organs and results in structural as well as functional deterioration of some tissues and organs. The study conducted by Leonard Hayflick in 1963 found that the frozen cells isolated from human will undergo deformation after been passaged for a certain number of times, the. Base on the results, human cell exist a upper limit for cell division, and the rate of cell division and the appearance of the cells will change after 50 passages, resulting in irregular division and granular- or twisted abnormal cell appearances and eventually inducing apoptosis of the cells. This study suggested that the life cycle of an organism was determined at the stage of fertilization and the biological clock of an organism was pre-determined. In humans, cells can divide up to 50 times which is around 120 years. Because human cells do not express telomerase for replication of the lost sequences during DNA replication and consequently genes are damaged over the processes of multiple replications and divisions and thus creates a restriction on cell cycle and such restriction is called glass ceiling and this is an irreversible and inevitable aging process.
(2) Ultraviolet (UV) Light
The skin and other organs will age over time, but the skin aging is more serious when compared with other organs due to the effects of various environmental factors in daily life. Among numerous aging factors, ultraviolet (UV) irradiation is the most influential factor of all. The UV wavelength ranges from 10 to 400 nm with energy between 3 eV and 124 eV. UV light can be divided into three classes based on its wavelength: (1) long-wavelength UV-A, with a wavelength between 315 and 400 nm, can directly pass through the atmosphere and reach the surface. It also can penetrate the dermis layer of the skin and cause black spots, aging and wrinkles UV-A posses the strongest penetration capacity among the 3 wavelengths; (2) medium-wavelength UV-B, with a wavelength between 280 and 315 nm, will result in skin redness, heat, pain, and even peeling or burns-like symptoms. But UV-B will be absorbed by stratospheric ozone and only limited irradiation will reach the surface of the Earth; (3) short-wavelength UV-C, with a wavelength between 100 and 280 nm, is the highest energy and is harmful. However, its short wavelength will be absorbed by the atmosphere and thus only 0.1% can reach the surface of the Earth. Moreover, general shield and glass barrier can block UV-C rays effectively. Previous study reported that UV-A may induce synthesis of matrix metalloproteinases in human skin fibroblasts and the members of the matrix metalloproteinases family will degrade collagen, elastin and other substances in intracellular matrix and cause aging. In addition, UV-A may increase the level of free radicals in cells and excess free radicals will also lead to premature aging or even apoptosis.
(3) Free Radical
Free radical theory is the most accepted scientific theory of aging at present and is proposed by Dr. Denham Harmam M.D. at Lincoln University School of Medicine in 1954. But the theory was not accepted until 20 years later and now it has become one of the major theories of aging. Denham Harmam was nominated and awarded the Nobel Prize in Medicine in 1995. Normal atoms contain paired electrons, whereas free radicals contain oxygen with unpaired electrons. Because unpaired electrons are extremely unstable, free radicals will transfer the electrons from normal atoms and thus induce change in the intracellular matrix and result in cell death.
In addition to metabolism or synthesis of nutrients in vivo, important sources of free radicals which can lead to aging also include environmental pollution, ultraviolet light, radiation, smoke, pesticides, and many chemicals, particularly environmental pollution (automobile exhaust and SO2 emissions by factories) and all these may contribute to production of significant amount of free radicals in vivo.
Free radical attack can be divided into cell membrane damage and DNA damage. For human cells, oxygen-containing free radicals include superoxide anion O2—, hydrogen peroxide H2O2, hydroxyl radical OH—, etc. and they are called reactive oxygen species (ROS). Excess free radicals will easily attack unsaturated fatty acids in cell membrane. When free radicals attack unsaturated fatty acids in cell membrane, lipid peroxidase will be produced and oxidize LDL cholesterol in vascular walls and inhibit the activity of prostacycline synthetase, resulting in atherosclerosis, diabetes, arthritis, cataracts, aging and coronary artery disease. If the free radical is deep down to the nucleus and altered genetic information, cancer may occur. In addition, free radicals can induce aging genes and promote aging. Studies have indicated that nearly 80%-90% of the aging and degenerative diseases are associated with free radicals, which include cancers, Alzheimer's disease, Parkinson's disease, muscular dystrophy, skin spot deposition, wrinkle formation, yellow spots, degenerative heart disease, stroke, ulcers, rheumatoid arthritis and multiple sclerosis.
Cellular defense mechanisms against free radicals: various antioxidant enzymes synthesized by the cells, e.g. enzymes glutathione Peroxidase (GPx) and superoxide Dismutase (SOD), and Glutathione (GSH), which can remove superoxide anions (2O2—+2H+H2O2+O2, 2GSH+H2O2 and GS—SG+2H2O) generated naturally in vivo. Studies have also suggested long-lived animals contain higher level of SOD in vivo and humans are the animals known by far that contain the highest level of SOD. Natural aging, physical changes and environmental factors can all cause insufficient production of antioxidants in vivo and result in aging.
(4) Inflammation
Inflammation is the response produced when the tissue is injured or infected. At first, the mast cells will arrive at the tissue and attach to the endothelial cells before releasing following substances:
(1) Histamine: a derivative of amino acid which can increase the permeability of capillaries and local vasodilation to allow substances such as plasma and macrophages to pass through and cause itching and allergic reactions.
(2) Tumor Necrosis Factor (TNF): cytokines will kill target cells and activate the immune system to induce proliferation of lymphocytes and prevent pathogen proliferation and also recruit macrophages to come to the site of infection.
(3) Prostaglandin: induce vasodilation of the capillaries and cause pain at the nerve ending. Pus will be produced following inflammation, which consists dead cells and body fluids and usually be digested by macrophages.
Specific cytokines will activate cyclooxygenase (COX), including COX-1 and COX-2. COX converts arachidonic acid into prostaglandins such as PGE 2 and PGF 2α. Recent studies have shown COX-2 is not found in most normal tissues, but it can be detected in patients with various cancers, indicating the importance of COX-2 in cancer patients. Additionally, COX-2 is an inducible enzyme whose function is to activate macrophages or other cells and exists in the inflammatory tissues.
The inflammatory sites will generate heat which is mainly due to releasing of angiotensin or histamine. Some cells will release the inflammatory cytokine IL-17 (Interleukin-1 alpha), formerly known as interleukin, to activate leukotriene to against allergens and the cytokines also include chemokines Said chemokines will initiate the chemoattractant mechanism and interferon and terminate protein synthesis in host cells. On the other hand, growth factors and cytotoxic molecules may also be released for healing of the tissues. Secretion of the abovementioned substances will affect the surrounding areas and cause loss of intracellular matrix and result in aging.
From the four aging factors mentioned above, during the process of skin aging, proliferation of the fibroblasts in epidermis and dermis layers will be decelerated which consequently contributes to changes of the large molecules and structural proteins in intracellular matrix of the dermis layer and result in symptoms of aging including skin folds, thinning of the skin, dull skin, reduced skin elasticity and moisture level. Alternatively, UV light induces production of intracellular proteases to break down collagen and elastic fibers in the skin tissue. Additionally, UV light may also increase the levels of free radicals in the cells and lead to premature aging due to skin inflammation. Moreover, the skin will generate ROS after UV light exposure that will destroy cellular structures such as cell membranes, structural proteins and nucleic acids and eventually result in cancerous development and death of the skin cells as well as wrinkles due to loss of intracellular matrix.
2. Human Anti-Aging Proteins
Some studies have indicated that under the stimulation of the chemical substance resveratrol in wines or restriction of calorie, the sirtuins family will be activated and extends the cell cycle, whereas Sinclair from the Leonard Guarente lab at MIT found that a special Sirtuins protein in yeast can affect the process of aging by using two specific approaches and Sirtuins can help regulate the gene activity of the cell and repair DNA breakage.
Philipp Oberdoerffer from the Sinclair lab, on the other hand, utilized microarrays of mouse cells to screen for the DNA sequence of mammalian sirtuin gene using the sirtuin gene from yeast, and the animal studies using mice also confirmed the yeast sirtuin-like gene exists in vertebrates as well. Oberdoerffer reported that the main function of Sirtuin in mammalian animals is to supervise the mode of gene expression. All genes will present in all cells; however, only limited genes need to be activated at specific time periods. If wrong genes were activated, cell damage will occur and induce apoptosis of the damaged cell.
Inhibited genes will undergo deacetylation induced by Sirtuins so as to protect the suppressed genes from damage resulting from environmental factors and ensure the suppressed genes remain shut down and safeguard the stability of the genes. Sirtuins can help preservation of chromatin and contract and cover the genes and histones and assure it remain idle. When the DNA is damaged by UV light or free radicals, Sirtuins will assist the repair mechanism at the damaged sites. Sirtuins covers the genes and proteins to exerts its protective function before permanent damage was resulted. Without the protection from Sirtuins, histones will start to relax the structure and the suppressed genes will be reactivated which makes the genes susceptible to external interferences and be damaged.
When the mice age, DNA damage rate will increase and this type of damage will lead to uncontrolled gene expression and relaxing of the chromatin. At the time, Sirtuins will help to control the deteriorated genome. Many of the genes activated during the loss-of-control process are the genes directly associated with the phenotypes of aging.
Other research suggested the mouse genes which were not controlled by Sirtuins will be continuously expressed in the aged mice. Oberdoerffer used a transgenic lymphoma mouse model to investigate the function of sirtuin and found that the average life span of the mice was extended 24%˜46% if extra copies of the sirtuin gene was given or the mice was fed with the sirtuin activator, resveratrol.
Alternatively, study conducted by Leonard Guarente indicated utilization of new drugs rearranged the distribution of Sirtuins over time and new approach will be generated to protect the cells from aging. Based on this specific mechanism, though DNA damage may deteriorate the process of aging, this result is not due to DNA damage, but, is resulted from lack of gene regulation. Furthermore, Oberdoerffer's study also showed that this process of regulation of gene expression is called epigenetics which is different from the actual mutation of DNA. By validation of this principle, it is found that stimulation of Sirtuins can reverse the process of aging.
3. Transdermal Penetration Enhancers (TPE)
Studying of new drug requires significant amount of money and time and therefore development of drug delivery systems has attracted more attention over the years. The most common drug administration methods include oral administration, subcutaneous injection and transdermal administration. Oral administration is the most common method of administration and the drug enters bloodstream after being absorbed by gastrointestinal mucosa and exerts its effect either locally or systematically. Nonetheless, the disadvantages of oral administration include slow and irregular absorption in vivo which reduces the treatment effects. Moreover, the drug has to pass through the liver before arrive at the blood which not only reduces the therapeutic effects of the drug but also increases the burden on the liver. Certain drugs cannot be orally administered due to poor intestinal absorption or irritation, and the most serious drawback of oral administration is the side effects of the drug which may cause discomfort in patients such as nausea and vomiting. Another administration method is subcutaneous injection which directly inject the drug into the subcutaneous region to allow absorption by subcutaneous capillaries and delivery to the body. The advantage of this method is the drug is not affected by gastric juice and the liver and can enter blood vessels directly and pass through various body parts and achieve the therapeutic effect. The subcutaneous injection significantly increases the rate of administration when compared with oral administration. However, for patients requiring long-term injections, it is a burden to bear the pain caused by prolonged injection. The other administration method is using the transdermal drug delivery system (TDDS) and the drug is absorbed by the skin in this system. After administration, the drug will pass through stratum corneum at pre-determined time periods and be absorbed by capillaries before entering blood circulation and exert its effect so as to achieve the aim of systematic therapy (Saunders et al., 1999).
The advantages of transdermal drug delivery system (TDDS) include easy production, low cost, constant rate for delivery, maintain long-term stable concentration of drug, lower administration frequency, low toxicity, reduced hepatic first pass effect, reduced drug metabolism, less individual differences in drug use, increased bioavailability and the therapeutic effects can be achieved with low dose administration. In addition, TDDS is suitable for used in children, elderly or patients with problems of drug intake; can be easily applied and be removed immediately to stop administration when problems occur. Because TDDS has the abovementioned advantages, this system has attracted much attention. At present, research and development of the TDDS system has evolved from local to systematic, target organs and controlled release and has been applied in clinical use (Shin et al., 2005).
The major obstacle of TDDS system is the skin's stratum corneum. Stratum corneum (SC) is the top layer of skin and is composed of flat and long keratin cells and surrounded by layers of fat (Norlen, 2001). The major function of SC is to prevent entry of foreign substances and water loss and is the outermost barrier of the skin (Bouwstra et al., 2003). In 1973, Breathnach et al. discovered the intracellular space between the cells in SC is filled with fat which plays an important role in the skin barrier function (Breathnach et al., 1973). Similarly, other studies also suggested that the fluidity of the fat in the intracellular space in SC increases when the temperature of SC elevates and consequently percutaneous absorption of the skin increases accordingly (Golden et al., 1987). The hydrophilicity and skin permeability of the amphipathic drugs increase significantly if the fat was removed from the SC; however, removal of fat showed no significant effect on lipophilic drugs (Tsai et al., 2001).
Three methods are available to overcome the obstacle of percutaneous absorption: first, the physical method, provide extra energy to create transient holes in the skin to promote drug absorption and common treatments include ultrasound, iontophoresis, microneedle array and thermal energy. The second approach is a biochemical method of using the combination of biotransformation of precursors and metabolic inhibitors to increase absorption of drugs. The third method is a chemical method in which liposome is used to cover the drugs or a penetration enhancer is added. Liposome consists of curved lipid bilayer with the hydrophilic end extruding outside and hydrophobic end facing inside and can be used as a carrier for both hydrophilic and hydrophobic substances. Oily drugs can integrate into the lipid bilayer, whereas hydrophilic drugs can be covered in the water phase of the liposomes. The interaction between liposome and cells involves four mechanisms: intermembrane transfer in which part of the composition of liposome is exchanged with the composition of the cell membrane; adsorption in which liposomes adhere to the cell membrane; fusion in which liposomes fuse with the cell membrane and deliver the content into the cell; and finally, endocytosis in which liposomes are taken by the cell. The penetration enhancer refers to substances which can promote permeability and the amount of drugs absorbed by the skin but will not cause serious irritation and damage (Williams and Barry, 1991). The penetration enhancer mainly take effect in the intercellular layer of the SC and disrupts its regular structure and increases fluidity; moreover, it acts on keratin to lossen the structure of keratinocytes, increase the solubility of drugs in SC and enhance absorption of drugs (Walker and Smith, 1996). Addition of penetration enhancer is a very helpful method to increase skin permeability (Saunders et al., 1999).
Surfactants are excellent skin penetration enhancers and can aid in increasing the permeability of biofilm and the skin (Lopez et al., 2000) and have been widely applied in drug penetration recently (Nokhodchi et al., 2003; Shokri et al., 2001). In 2001, Nokhodchi et. al suggested surfactants sodium lauryl sulfate (SLS), cetyltrimethylammonium bromide (CTAB) and benzalkonium chloride can enhance skin absorption of the anti-depression drug Diazepam in mice (Shokri et al., 2001). Another study conducted by Nokhodchi et. al on anti-depression drugs also indicated surfactants sodium lauryl sulfate (SLS), cetyltrimethylammonium bromide (CTAB) and benzalkonium chloride can enhance skin absorption of lorazepam in mice (Nokhodchi et al., 2003). Surfactin exhibits great affinity for synthetic cell membranes, prokaryotic cell membranes and eukaryotic cell membranes (Maget-Dana and Ptak, 1995; Sheppard et al., 1991; Tsukagoshi et al., 1970b) and the binding between surfactin and cell membranes is highly selective which is due to surfactin has high affinity for cholesterol and phospholipid and these two structures are the major constituents of the cell membrane (Hosono and Suzuki, 1985). When compared with chemically synthesized surfactants, surfactin is more gentle and does not harm to the skin.
By conventional methods, drugs or nutritional active substances can only penetrate the barrier of epidermis (stratum corneum) and the exerted effects are not significant (up to 0.3% effectiveness). To solve this problem, a number of TDDS systems have been developed for the purpose of enhancing nutrient penetration so as to allow the nutrients to pass through epidermis and dermis layers of the skin and consequently nutrient delivery method has become the major research topic of skin care technology.
Although the stratum corneum of human skin is very thin, only 10-25 microns in thickness, and the thinnest SC is at the cuticle eyelids, 6 microns, the SC is rather “tough” and is the most important protective layer of the skin. Common cosmetics primarily penetrate the skin through 3 major pathways: 1. through sweat duct; 2. pas through stratum corneum directly; and 3. through hair follicle.
Using the cosmetic ingredient gold as an example, modern studies on gold beauty has confirmed that gold has the functions of detoxification, calm, clean and wrinkle reduction and can rearrange cellular factors, promote physiological functions and metabolism, balance oil/water secretion, retain natural water and prevent allergy caused by external factors. One the other hand, nanogold, a common ingredient used in cosmetic products has a size close to 1/200 of a human pore and thus the purpose of using skin care products containing nano-gold particles is to facilitate penetration of nanogold to the cells in deep dermis.
After entering the dermal layer, nanogold can regulate the function of dermal cells at the genetic level, including induce dermal cells to produce a series of active substances such as SOD, metallothionein and EGF. Because SOD can scavenge hydroxyl radicals, and metallothionein can help cortical cells to resist UV light damage, nanogold particles posses the anti-aging effect in dermal cells. Other studies also suggested nanogold can induce fibroblasts to secret and synthesize extracellular matrix (ECM) and to express and secret epidermal growth factor (EGF) in order to different target cell-specific keratinocyte growth factor (KGF) while strengthening the firmness of the skin and make the skin smooth and full of elasticity with shining glory.
4. Bioemulsifiers
In addition to be used as an anti-bacterial peptide, surfactin also plays another important role, bioemulsifier. Deleu et. al in 1999 discovered that ituirn A has better effect than surfactin in creaming-flocculation inhibition tests, while surfactin posses superior effect in emulsification of alkanes when compared with ituirn A and fengycin, and SDS was found to have the least effect in emulsification. Other research has shown that addition of 20 mg/l of surfactin can increase the biodegradability of diesel and further indicated pH value will affect the emulsification effect of surfactin on diesel and surfactin has the best biodegradability effect on diesel when the pH was adjusted to 7.4.
5. Foaming Agents
Besides the emulsification capability of common emulsifiers, surfactin also has the capacity to facilitate formation of foam (Razafindralambo et al., 1998). The foaming effect refers to surfactin exists between the gas and liquid phase and vigorous shaking allows surfactants to grasp the air and form a thin film containing air (Halling, 1981). Razafindralambo et. al also suggested surfactin presents better foaming effect when compared with ituirn A and speculated that the structures of these two agents are related to their foaming properties. Surfactin belongs to the anionic surfactants and its fatty acid carbon chain is shorter, while ituirn A is a non-ionic surfactant with a longer fatty acid chain (Razafindralambo, et al., 1998).